The present invention relates genetically modified cells, to an assay and methods and the usage thereof to measure the infectivity and viral resistant/sensitivity of isolate from peripheral blood mononuclear cells (PBMC) and plasma of an immunodeficiency virus. The present invention has utility in determining the HIV co-receptor usage, discovery of new drugs effective against HIV and monitoring a drug therapy protocol in order to enhance the effectiveness of drug treatment regimes against HIV-1 infection,
There is currently no cost effective, xe2x80x9chigh throughputxe2x80x9d method for analyzing the drug resistant phenotype of primary virus isolates derived from individuals receiving antiretroviral treatment. Various in vitro biologic and immunologic techniques have been developed to detect human and simian immunodeficiency viruses (HIV and SIV, respectively). These include assays that detect the enzymatic activity of the reverse transcriptase (RT) protein, ELISA based assays for the detection of HIV/SIV core antigen (HIV-1 p24 or HIV-2/SIV p27), direct quantitation of infectious virus by syncytial focus plaque assays or limiting dilution titration in susceptible host cells, visualization of virions by electron microscopy, in situ hybridization, and various nucleic acid-based assays. Recently, genetic reporter-based assays have been created to detect HIV/SIV infection. In this approach, mammalian cells are genetically modified to express a reporter gene such as xcex2-galactosidase (xcex2-gal), green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT) in response to infection and Tat protein expression. These detection systems require enumeration of the number of infection-positive cells by flow cytometry or fluorescence microscopy (GFP), microscopy (xcex2-gal), or the utilization of radioisotopes (CAT). The firefly luciferase gene, under control of the HIV-1 LTR promoter, has been used as a reporter gene for HIV-1 infection. Luciferase is very sensitive marker gene for HIV-1 infection, since expression of a relatively few number of luciferase molecules can result in appreciable activity levels using standard luciferase detection assays.
The sensitive detection of the virus quasispecies that comprise primary HIV isolates has proved difficult using immortalized CD4 positive cell lines. At least in part, this has been due to the lack of expression of the CCR5 chemokine co-receptor on the surface of such cell lines. The failure to detect infection of primary virus isolates (T-cell and macrophage tropic viruses) using immortalized cell lines has greatly impeded the development of useful approaches for detecting, quantifying and analyzing HIV infection of primary virus isolates. The present invention largely overcomes the prior art limitations.